Diabetes Page 2 of 47
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Hepatic cyclooxygenase 2 expression protects against diet induced steatosis, obesity and insulin resistance. ●1Daniel E. Francés, ●2Omar Motiño, ●2Noelia Agrá, 2,3Águeda González Rodríguez,
1Ana Fernández Álvarez, 5Carme Cucarella, 4,6Rafael Mayoral, 2Luis Castro Sánchez,
2Ester García Casarrubios, 2,4Lisardo Boscá, 1Cristina E. Carnovale, 4,5Marta Casado,
♦2,3Ángela M. Valverde and ♦2,4Paloma Martín Sanz.
Keywords: COX 2, hepatocyte, HFD, obesity, IR.
Running title: COX 2 in insulin resistance
Affiliations:
1Institute of Experimental Physiology (IFISE CONICET), Suipacha 570, 2000 Rosario,
Argentina. 2Institute of Biomedical Research Alberto Sols, CSIC UAM, Arturo Duperier
4, 28029 Madrid, Spain; 3Networked Biomedical Research Center on Diabetes and
Metabolic Diseases (CIBERdem), Monforte de Lemos 3 5, 28029 Madrid, Spain;
4Networked Biomedical Research Center on Hepatic and Digestive Diseases
(CIBERehd), Monforte de Lemos 3 5, 28029 Madrid, Spain; 5Biomedical Institute of
Valencia, IBV CSIC, Jaume Roig 11, 46010 Valencia, Spain; 6Department of Medicine,
University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
♦ Corresponding authors.
These two authors share senior authorship (♦)
Dr. Paloma Martín Sanz or Dr. Ángela M. Valverde, Institute of Biomedical Research
Alberto Sols, CSIC UAM. Madrid. Arturo Duperier, 4 28029 Madrid, Spain Tel
34914972746, 34915854497 Fax: 34915854401; e mail: [email protected] or [email protected]
● These authors contributed equally to this work
Diabetes Publish Ahead of Print, published online November 24, 2014 Page 3 of 47 Diabetes
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Figures: 5
Tables: 3
Word count abstract: 198
Word count manuscript: 3865
Abbreviations
COX 2, cyclooxygenase 2; PGE2, prostaglandin E2; RCD, regular chow diet; HFD, high fat
diet; Ins, Insulin; DFU,(5,5 dimethyl 3(3 fluorophenyl) 4 (4 methylsulfonyl) phenyl 2(5H)
furanone); PTP1B, protein tyrosine phosphatase 1B; Cpt1a, carnitine palmitoyltransferase 1
liver isoform; Scd1, stearoyl CoA desaturase 1, TG, triglycerides; Pgc1 α, PPARγ coactivator
1α; Acox1, palmitoyl CoA oxidase; Ppar α, peroxisome proliferator activated receptor alpha;
Ppar γ, peroxisome proliferator activated receptor gamma; Acaca, acetyl CoA carboxylase 1;
Fasn, fatty acid synthase; Lipc, hepatic triacylglycerol lipase; Lipe, hormone sensitive lipase;
Pnpla2, patatin like phospholipase domain containing protein 2; Cd36, fatty acid translocase;
mpges 1, microsomal prostaglandin E synthase 1; Bcl 2, B cell lymphoma 2; UCP 1,
uncoupling protein 1; Dio2, type II iodothyronine deiodinase; Prdm16, PR domain containing
16; ALT, alanine transaminase; AST, aspartate transaminase, NEFAs, non esterified free fatty
acids; HOMA, homeostasis model assessment of insulin resistance; IR, insulin resistance; IL 6,
interleukin 6; IL 1β, interleukin 1β; TNF α; tumor necrosis factor α; EE, energy expenditure;
RER, respiratory exchange ratio; SVF, stromal vascular fraction.
Diabetes Page 4 of 47
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ABSTRACT
Accumulation evidence links obesity induced inflammation as an important contributor to the development of insulin resistance which plays a key role in the pathophysiology of obesity related diseases such as type 2 diabetes mellitus and non alcoholic fatty liver disease. Cyclooxygenase (COX) 1 and 2 catalyze the first step in prostanoid biosynthesis. Since adult hepatocytes fail to induce COX 2 expression regardless of the pro inflammatory stimuli used, we have evaluated whether this lack of expression under mild pro inflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX 2
(hCOX 2) in hepatocytes protects against adiposity, inflammation, and hence insulin resistance induced by high fat diet as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin/leptin ratio and decreased levels of pro inflammatory cytokines together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX 2 transgenic mice exhibited increased whole body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptor mediated Akt phosphorylation in hCOX 2 Tg. In conclusion, our results point to COX 2 as a potential therapeutic target against obesity associated metabolic dysfunction.
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INTRODUCTION
Insulin resistance (IR) plays a key role in the pathophysiology of obesity related
diseases such as type 2 diabetes and non alcoholic fatty liver disease (NAFLD). IR is
the key primary defect underlying the development of type 2 diabetes and is a central
component defining the metabolic syndrome. It has been demonstrated that IR is
associated with a state of chronic low grade inflammation, and it is assumed that
inflammation contributes in a major way to its development (1). Besides the fact that IR
is characterized by complex interactions between genetic determinants and nutritional
factors, it is recognized that mediators synthesized from immune cells and adipocytes
are involved in the regulation of insulin action (2). Insulin acts in target cells by binding
to its specific receptor and activating a cascade of intracellular signaling events that are
altered in inflammation associated IR (3).
Inflammation induces the expression of a variety of proteins including
prostaglandin endoperoxide synthase 2, also known as cyclooxygenase (COX) 2, which
catalyzes the first step in prostanoid biosynthesis. COX 2 is induced by a variety of
stimuli such as growth factors, pro inflammatory mediators, hormones and other
cellular stresses (4). However, adult hepatocytes fail to induce COX 2 regardless of the
pro inflammatory stimuli used and only under prolonged aggression as a result of the
drop of C/EBPα (5) or after partial hepatectomy (PH), COX 2 is induced (6). On the
other hand, Kupffer, stellate, hepatoma mouse liver cells and fetal hepatocytes retain the
ability to express COX 2 upon stimulation with LPS and pro inflammatory cytokines
(7).
Previous work has implicated COX 2 in the pathogenesis of obesity and IR.
Hsieh et al. (8 10) reported an improvement of muscle and fat insulin resistance in rats
fed with fructose or high fat diet (HFD) treated with COX 2 inhibitors. However, Coll Diabetes Page 6 of 47
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et al. found that COX 2 inhibition exacerbates palmitate induced inflammation and IR in skeletal muscle (11). Our previous results (12) performed in a model of transgenic mice constitutively expressing human COX 2 in hepatocytes (hCOX 2 Tg) demonstrated a protective role of COX 2 against liver apoptosis induced by streptozotocin (STZ) mediated hyperglycemia. These findings prompted us to screen the role of COX 2 expression in hepatocytes in a model of IR and altered energy homeostasis induced by HFD. The present study has demonstrated that expression of
COX 2 in hepatocytes protects against steatosis, adiposity, inflammation and hepatic insulin resistance, in hCOX 2 Tg mice under HFD by improving insulin sensitivity and glucose tolerance, enhancing Akt and AMPK phosphorylation, decreasing protein tyrosine phosphatase 1B (PTP1B) protein levels and increasing thermogenesis and energy expenditure.
RESEARCH DESIGN AND METHODS
Animal experimentation. hCOX 2 Tg mice (25 30g body weight; 3 months) on a B6D2/OlaHsd background were used in this study along with corresponding age matched wild type (Wt) mice (13).
Animals were treated according to the Institutional Care Instructions (Bioethical
Commission from Spanish Research Council, CSIC, Spain). hCOX 2 Tg mice and their corresponding Wt littermates were generated by systematic mating of Tg mice with
B6D2F1/OlaHsd Wt mice in our animal facilities for more than seven generations.
Insulin signaling studies. 6 h fasted Wt and hCOX 2 Tg mice were i.p. injected with
0.75 U/kg human recombinant insulin, and sacrificed 15 min later. Liver was removed, and RNA and total protein extracts were prepared as previously described (14).
Induction of liver steatosis and obesity by high fat diet (HFD). Wt and hCOX 2 Tg mice were fed with regular chow diet (RCD, A04 10, Panlab, Barcelona, Spain) or a Page 7 of 47 Diabetes
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42% high fat diet (HFD) (TD. 88137, Harlan Laboratories, Madison, WI, USA) ad
libitum for 12 weeks. Some (n=7) of the hCOX 2 Tg mice fed a HFD were treated i.p.
with 10 mg/kg /day DFU, a COX 2 selective inhibitor, five times in a week during all
the treatment. During HFD treatment, body weight, food and water intake were
recorded every two days. After 12 weeks of treatment, animals were sacrificed and
liver, epididymal WAT (eWAT), inguinal WAT (iWAT) and interscapular brown
adipose tissue (BAT) were snap frozen in liquid nitrogen and stored at 80°C or
collected in a solution containing 30% sucrose in PBS or fixed in 10% buffered
formalin. Plasma was obtained from the inferior vena cava.
Metabolic measurements. Metabolic measurements were performed at the beginning of
HFD treatment and at the end of week 12. Mice were subjected to insulin tolerance test
(ITT) and glucose tolerance test (GTT). For ITT, after 6 h fasting, mice were i.p.
injected 0.75 U/kg of human recombinant insulin. Blood glucose was measured from
the tail vein at 0, 15, 30, 60 and 90 min. For GTT, 16 h fasted mice were i.p. injected 2
g/kg glucose and blood samples were taken at 0, 30, 60 and 90 min. Glucose levels were
measured with an Accu Check Glucometer (Roche). The HOMA IR (homeostasis
model of assessment of insulin resistance), an index of whole body insulin resistance at
basal state, was calculated by HOMA IR=(FPIxFPG)/22.5; where FPI is fasting plasma
insulin concentration (mU/l) and FPG is fasting plasma glucose (mmol/l).
Indirect calorimetry. At the end of week 12, independent groups of Wt and hCOX 2 Tg
mice (n=4) were analyzed by indirect calorimetry in a PhenoMaster System (TSE
systems, Bad Homburg, Germany). Mice were acclimated to the test chamber for at
least 24 h and then were monitored for an additional 48 h. Food and water were
provided ad libitum in the appropriate devices and measured by the build in automated
instruments. Volumes of consumed O2 (VO2) and eliminated CO2 (VCO2) were taken Diabetes Page 8 of 47
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every 10 min. Respiratory exchange ratio was calculated as: RER=VCO2/VO2. Energy expenditure was calculated as: EE=(3.815 + (1.23 x RER)) x VO2 x 1.44). Data are the average values obtained in these recordings.
Cold exposure experiments. Three month old Wt and hCOX 2 Tg mice (n=8) were acclimated at a temperature of 28ºC for one week. Then, mice were randomly divided into two groups, one group was exposed to 4ºC for 6 h in individual cages and the other group was maintained at 28ºC. At the end of the experiment, mice were sacrificed and
BAT, eWAT and iWAT depots were removed and frozen in liquid nitrogen for posterior assays.
Data analysis.
Statistical analyses were performed using the SPSS package v.17. Data are expressed as means ± S.E. Student’s t test was applied whenever necessary, and statistical analysis of the differences between groups was performed by one way ANOVA followed by
Tukey’s test. A p<0.05 was considered statistically significant.
RESULTS
COX 2 transgenic mice are protected from HFD induced hepatic steatosis, obesity and insulin resistance.
To explore if COX 2 expression in hepatocytes affects glucose tolerance and insulin sensitivity in mice, we used our previously described transgenic mice (hCOX 2Tg) that constitutively express human COX 2 in hepatocytes under the control of the human
ApoE promoter and its specific hepatic control region (HCR), a unique regulatory domain that directs ApoE expression in liver (13), that also lacks macrophage specific regulatory regions (ME.2 and ME.1) (15), ensuring exclusive expression in liver. PGE2, the main COX 2 derived prostanoid in liver, is three fold increased (Supplementary Fig.
1A and B) (13). The expression of COX 2 in the liver of COX 2 Tg mice is comparable Page 9 of 47 Diabetes
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to the levels reached in fetal hepatocytes after stimulation with LPS (5; 7) and in liver
regeneration after PH (6; 16). Although, no differences in body weight gain were found
under RCD diet (Supplementary Fig. 1C), hCOX 2 Tg mice fed with a HFD diet did
not increase body weight as much as the corresponding Wt littermates and the eWAT
size and liver weight were significantly lower compared to those of the Wt (1.29±0.05*;
1.63±0.11 liver weight for hCOX 2 Tg and Wt respectively) (Fig. 1A C). Notably,
hCOX 2 Tg mice displayed a 20% lesser body weight gain without significant changes
in the initial body weight or in food intake (Fig. 1D). Interestingly enough, hCOX 2 Tg
mice treated with DFU exhibited an intermediate body weight value (Fig. 1C). To
assess the impact of HFD on glucose homeostasis, HFD fed mice were subjected to
GTT and ITT analysis (Fig. 1E F). Plasma glucose levels (mg/dl) were similar between
genotypes under HFD: 173.5±6.7; 159.2±12.1 and 177.3±21.2 for Wt, hCOX 2 Tg and
hCOX 2 Tg+DFU, respectively. However, under 42% HFD, hCOX 2 Tg mice
displayed enhanced insulin sensitivity (Figure 1E) and glucose tolerance (Figure 1F)
than the Wt. In addition, treatment of hCOX 2 Tg mice with DFU partially reversed the
beneficial effects on glucose homeostasis induced by COX 2 dependent prostaglandins.
ITT and GTT also revealed an enhanced insulin sensitivity and glucose tolerance in
hCOX 2 Tg vs. Wt mice under basal conditions (regular chow diet, RCD;
Supplementary Fig. 1D and E). The effect of COX 2 expression in hepatic insulin
signaling was evaluated in primary hepatocytes. As shown in Supplementary Fig. 2A,
insulin induced insulin receptor and Akt phosphorylations were markedly increased in
hCOX 2 Tg hepatocytes. Furthermore, when Wt hepatocytes were treated with
prostaglandin E2 (PGE2) prior to insulin stimulation Akt phosphorylation was increased
and, conversely, when hCOX 2 Tg hepatocytes were pretreated with DFU, Akt Diabetes Page 10 of 47
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phosphorylation was decreased, indicating the specificity of COX 2 dependent prostaglandins in the modulation of insulin signaling (Supplementary Fig. 2B).
Chronic HFD treatment causes accumulation of lipids in the liver, a process leading to fatty liver disease. Under these nutritional conditions Wt mice developed severe steatosis (Fig. 2A), including massive accumulation of large lipid droplets and a significant increase in liver TG (Fig. 2B). hCOX 2 Tg mice were protected against liver steatosis, showing lesser and smaller lipid droplets whereas DFU administration partially reverted this effect exhibiting an intermediate liver steatosis but including lipids microvesicles (Supplementary Fig. 3A); steatosis score as defined by NASH
Clinical Research Network Scoring System Definitions (17) was lower in hCOX 2 Tg mice (Supplementary Fig. 3B).
COX 2 transgenic mice are protected from hepatic inflammation and injury.
Since the increase in adipose tissue and inflammation has been linked to insulin resistance, we performed histological examination of eWAT and the results showed adipocyte hypertrophy with larger adipocytes in Wt mice vs. hCOX 2 Tg and, again,
COX 2Tg mice treated with DFU resembled features of the Wt eWAT (Fig. 2C). A frequency histogram showed a shift to the left in adipocyte size in the hCOX 2 Tg mice with a significant increase in the number of cells and a decrease in the occupied area
(mean 1168±652, 624±356* for Wt and hCOX 2 Tg respectively); this process was partially reverted with the DFU treatment (748±863) (Fig. 2D and E). Moreover, as an estimation of body fat content, eWAT plus iWAT mass referred to body weight was lower in hCOX 2 Tg mice whereas tibia length did not change between Wt and hCOX
2 Tg mice (Fig. 2F). These results indicate hypertrophy in eWAT from Wt mice.
Cytokine eWAT expression is shown in Fig. 2G. As expected, higher adiponectin levels Page 11 of 47 Diabetes
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were detected in hCOX 2 Tg mice together with a significant decrease in the pro
inflammatory markers TNF α and IL 6.
Indeed, whereas plasma TGs were significantly elevated exclusively in obese Wt mice
after HFD, plasma cholesterol increased both in Wt and hCOX 2 Tg mice. Regarding
plasma free fatty acids (NEFA) levels, those in HFD fed Wt mice were higher than in
hCOX 2 Tg mice (Table I). Plasma insulin and leptin increased after HFD in Wt mice,
but lesser increases were found in hCOX 2 Tg mice. Furthermore, hCOX 2 Tg mice
were hypoleptinemic under RCD (Table I), and adiponectin levels were elevated (20%
increase) in hCOX 2 Tg mice vs. Wt controls under HFD. The adiponectin/leptin ratio
was higher in hCOX 2 Tg; however, HOMA IR was lower in hCOX 2 Tg mice under
HFD (Table I). hCOX 2 Tg mice treated with DFU partially prevented the beneficial
effects on plasmatic parameters induced by COX 2 dependent prostaglandins. In fact,
plasmatic PGE2 did not change with HFD however, as expected, PGE2 levels decreased
under DFU treatment (Table I). Hepatic injury was analyzed by measuring ALT and
AST activities and both enzymes increased by the effect of HFD to a lesser extend in
hCOX 2 Tg mice (Supplementary Fig. 4). HFD led to a chronic inflammatory profile,
which is believed to be critical for the development of glucose intolerance and insulin
resistance (18). Accordingly, hepatic mRNA levels of TNF α, Il 1β and IL 6, were
significantly enhanced in livers from HFD fed Wt mice, whereas hCOX 2 Tg mice
exhibited lower levels of these inflammatory markers (Table II).
After detecting the differences in NEFAs levels, High Resolution Magic Angle
Spinning Magnetic Resonance Spectroscopy (HR MAS MRS) was performed to
identify and quantify saturated fatty acids (Supplementary Fig. 5A and B). We found a
lower value of the pool of total saturated fatty acids in hCOX 2 Tg mice compared to
Wt, and this result was reversed in hCOX2 Tg+DFU (Supplementary Fig. 5B). Diabetes Page 12 of 47
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To gain insight into the molecular mechanisms implicated in COX 2 protection, we evaluated mRNA levels of transcription factors and genes involved in fatty acid oxidation, lipolysis and lipogenesis. In hCOX 2 Tg liver there was a significant increase of Ppara and Cpt1a under HFD indicating an enhanced fatty acid oxidation. In agreement, the expression levels of genes involved in fatty acid synthesis such as Pparg and Scd1 were markedly increased in Wt, but up regulation of these markers was lower in hCOX 2 Tg mice. Also, Cd36 expression was lower in hCOX 2 Tg. Moreover, gene expression of COX 2 mediated responsive primary targets has been analyzed. As expected, hCOX 2 Tg mice showed an increase in hepatic mRNA levels of CyclinD1 and Bcl2, known target genes of COX 2, and mPges1, the second key enzyme that couples with COX 2 for the synthesis of PGE2 (Table III).
Increased energy expenditure in COX 2 transgenic mice after HFD treatment.
To determine the cause of decreased adiposity and improved insulin sensitivity in hCOX 2 Tg mice, we examined food intake and energy expenditure (EE). The reduced adiposity of hCOX 2 Tg mice could not be explained by decreased food intake (Fig.
1D), but was associated with increased EE. To note, no differences were found in basal rectal temperature by a possible pyrogenic effect of PGE2 (Wt: 36.4±0.6; h COX 2 Tg:
35.8±0.7). Oxygen consumption (VO2) was elevated in hCOX 2 Tg mice vs. Wt mice during light and dark cycles. As expected, DFU treatment of hCOX 2 Tg resembled the
Wt situation (Fig. 3A and Supplementary Fig. 6A). Similar results were obtained when
CO2 production was measured and as consequence, EE and RER were also increased in hCOX 2 Tg mice vs. Wt mice during light and dark periods (Fig. 3B, 3C, 3E and
Supplementary Fig. 6E). However, no differences between genotypes were noted in locomotor activity (Fig. 3D). In agreement with increased cage temperature (Fig. 3F), hepatic COX 2 derived PGE2 enhanced the expression of thermogenic genes (Ucp1, Page 13 of 47 Diabetes
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Dio2 and Prdm16) in iWAT, suggesting induction of browning. Furthermore,
thermogenic gene expression was also increased in BAT in hCOX 2 Tg mice (Fig. 4A).
To reinforce these data, we performed a cold acclimation experiment to assess the
differential thermogenic response between Wt and hCOX 2 Tg mice. As shown in
Figure 4B, cold induced Ucp1, Dio2 and Prdm16 mRNA levels were significantly
higher in iWAT of hCOX 2 Tg mice, suggesting a browning process. In BAT, the
differential effects after cold exposure were found in Ucp1 mRNA levels. These data
reinforce the beneficial effects found in energy expenditure in hCOX 2 Tg mice. Also,
PGE2 was able to induce UCP 1 expression in non differentiated BAT SVF cells (Fig.
4C).
Hepatic COX 2 expression enhances insulin signaling in liver after HFD.
The phosphorylation of Akt and AMPK α were markedly reduced in obese Wt liver as
compared to hCOX 2 Tg mice (Fig.5A, 5B). These differences in insulin signaling
between Wt and hCOX 2 Tg mice after HFD could reflect differential expression of
negative modulators of the early steps of insulin signaling. In this regard, PTP1B
protein levels were up regulated in the liver of HFD fed Wt mice compared to similar
genotype fed with RCD, whereas this effect was not observed in hCOX 2 Tg mice. All
these data suggest that hCOX 2 Tg mice are protected against insulin resistance under
HFD.
Human and mouse liver cells expressing a COX 2 Tg are protected against fatty acids
exposure.
To confirm whether liver cells expressing COX 2 exhibit a higher insulin sensitivity
and are protected against IR, CHL (human) and NCL (murine) cells with (CHL C,
NCL C) or without (CHL V, NCL V) COX 2 expression were treated with 600 µM of
palmitate for 16 h and then stimulated with 10 nM insulin for 15 min. As shown in Diabetes Page 14 of 47
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Supplementary Fig. 7A C, CHL C and NCL C cells per se showed a higher increase in
Akt phosphorylation in response to insulin than CHL V and NCL V cells. Moreover, when cells were treated with palmitate to induce IR, CHL C and NCL C exhibited a higher Akt phosphorylation in response to insulin than CHL V and NCL V reinforcing the results obtained in vivo in hCOX 2 Tg mice.
DISCUSSION
The present study has demonstrated that constitutive expression of COX 2 in hepatocytes protects against steatosis, adiposity, inflammation and hepatic insulin resistance in mice under HFD, implicating the lack of COX 2 expression under these circumstances as a novel key player in the development of obesity associated metabolic dysfunction. Importantly, the leanness of hCOX 2 Tg mice is associated with increased systemic EE, enhanced thermogenesis and fatty acid oxidation in the liver. Furthermore, pharmacological inhibition of COX 2 with DFU reverts to the situation of the Wt mice.
Our previous results (12), by using this transgenic model, demonstrated a protective role of COX 2 against liver injury in an experimental model of STZ mediated hyperglycemia. The present study reveals that hCOX 2 Tg mice exhibit increased insulin sensitivity both in basal conditions and after HFD and are protected against HFD induced glucose intolerance, steatosis and adiposity. Recent studies suggested that COX 2 expression in eWAT protects against obesity by an increase in thermogenic activity (19). Moreover, this effect has been attributed principally to COX
2 derived PGE2 (20). In agreement with this, the reduced adiposity found in hCOX 2
Tg mice under HFD could be explained as a consequence of elevated EE due to increased substrate consumption. In this way, circulating liver derived PGE2 might be sufficient for the induction of thermogenic genes in adipose tissues, all of these contributing to protection against HFD induced obesity and improvement of glucose Page 15 of 47 Diabetes
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homeostasis. This was supported by the enhanced thermogenic gene expression found
in iWAT and BAT of hCOX 2 Tg mice under HFD or cold exposure and by the
induction of UCP 1 protein levels in brown preadipocytes stimulated with PGE2.
Elevation of circulating fatty acids and pro inflammatory adipokines secreted by
WAT reduces insulin sensitivity in liver and skeletal muscle (21) and adipose tissue
inflammation has been correlated with hepatic steatosis in humans (22). Our results
show that hCOX 2 Tg mice did not develop the common increase in fat mass and
enhanced adipocyte hypertrophy that is shown in Wt mice under HFD. In agreement
with these data, plasma and hepatic TGs, cholesterol and NEFAs levels were lower in
hCOX 2 Tg mice and also these mice showed decreased plasma insulin and leptin and
increased adiponectin levels. Moreover, pro inflammatory markers were also lower in
hepatic tissue from hCOX 2 Tg mice vs. with Wt after HFD. Altogether, these
parameters indicate that the beneficial effects on obesity and glucose homeostasis in
hCOX 2 Tg mice might be due to decreased inflammation.
When Wt and hCOX 2 Tg mice were fed a HFD we found an important
protection against steatosis and IR vs. Wt mice. Moreover, we found significant
differences in the expression of key enzymes and transcription factors implicated in
lipogenesis and β oxidation, such as Pparg, Scd1, Ppara, Cpt1a and CD36 indicating an
increase in β oxidation in hCOX 2 Tg mice under HFD. Recent results have implicated
hepatic fatty acid translocase CD36 upregulation with IR and steatosis in non alcoholic
steatohepatitis and chronic hepatitis C (23). In addition to this, the positive effects of
COX 2 on inflammation and IR are evidenced by the prevention of obesity through
increased EE and RER. Diabetes Page 16 of 47
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The PI3K/Akt and AMPK pathways play a central role in integrating diverse survival signals. Moreover, Akt phosphorylation is a critical node of the insulin signaling pathway, being tyrosine phosphorylated IRS 1 an upstream mediator of its activation (1). It is known that the Akt pathway is a target of PGs and that Akt phosphorylation is enhanced in the liver of hCOX 2 Tg mice compared to the Wt, thus reinforcing the survival pathways (12). In Wt liver, HFD impaired insulin stimulated
Akt phosphorylation and this effect was prevented in hCOX 2 Tg mice. This situation was paralleled in both murine and human derived liver cells overexpressing COX 2.
The increase in pAkt/Akt ratio in hCOX 2 Tg mice under RCD and HFD conditions may be due, besides intracellular signaling, to a direct Akt activation through COX 2 dependent PGE2 acting via EP2/EP4 Gβγ dimers as reported by Rizzo et al. (24).
Notably, the effect of PGE2 pretreatment on Akt phosphorylation observed in isolated hepatocytes confirmed that COX 2 derived prostanoids can be responsible of this effect.
Thus, the improved insulin sensitivity in vivo reflected by hepatic Akt activation found in hCOX 2 Tg mice might contribute to the amelioration of HFD derived deleterious metabolic effects as exacerbated lipogenesis and hepatic triglyceride content.
It is known that AMPKα1 protects mice from diet induced obesity and IR.
AMPKα1 knockdown in mice enhanced adipocyte lipid accumulation, exacerbated the inflammatory response and induced IR (25). We showed that AMPK was phosphorylated in liver cells expressing COX 2 under basal conditions and after liver injury (16). Indeed, AMPK phosphorylation and its downstream gene Cpt1a, which regulates a rate controlling step of fatty acid oxidation transferring long chain acyl CoA into the mitochondria, were increased in hCOX 2 Tg mice under HFD. Cellular lipid content is determined by the balance between fatty acid oxidation and lipid storage as Page 17 of 47 Diabetes
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TGs. Since the ability of AMPK to induce lipid oxidation is considered an important
feature for insulin sensitization (26), this might also contribute to the beneficial effects
of COX 2 expression in hepatocytes on insulin sensitivity.
In addition to the molecular mechanisms mentioned above, differences in insulin
signaling between Wt and hCOX 2 Tg mice after HFD could reflect differential
expression of negative modulators of the early steps of insulin signaling. Among them,
PTP1B was a likely candidate given its ability to directly dephosphorylate the insulin
receptor and also its expression is induced by inflammatory mediators (27). PTP1B KO
mice are protected against obesity induced inflammation and peripheral insulin
resistance during aging (28). In agreement with these data, PTP1B protein was up
regulated in the liver of HFD fed Wt mice but not in hCOX 2 Tg mice, suggesting that
COX 2 dependent prostaglandins might protect from the elevation of PTP1B during
obesity. In addition, PTP1B modulates leptin sensitivity (29) and decreased leptin levels
are found in hCOX 2 Tg mice in both RCD and HFD conditions suggesting a possible
cross talk between COX 2 derived signals and leptin.
Henkel et al. reported that PGE2 produced in Kupffer cells interrupt the
intracellular insulin signaling in hepatocytes through serine phosphorylation of IRS 1
via EP3 receptor dependent ERK1/2 activation. Moreover; they demonstrated that in
hepatocytes the EP3 receptor is expressed at higher levels that the other isoforms (30).
However, our results show that the liver of Wt and hCOX 2 Tg mice expresses
comparable levels of all the EPs receptors (EP1 EP4) (not shown) indicating a different
molecular mechanism of action. Our in vivo data in the hCOX 2 Tg model
characterized by a continuous production of PGE2 in the liver clearly show enhanced
insulin mediated Akt phosphorylation partly due to decreased PTP1B levels under Diabetes Page 18 of 47
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HFD. Therefore, the view emerging from this study is that PGs synthesized by other liver cells (Kupffer or stellate) are not sufficient to cope for the beneficial effects observed when hepatocytes are able to express COX 2. In conclusion, the present study demonstrates that expression of COX 2 in hepatocytes protects against adiposity, inflammation and hepatic IR in mice under HFD, pointing to COX 2 as a potential therapeutic target against obesity associated metabolic dysfunction. Stable analogues of
PGE2 such as 16,16dmPGE2, resembling COX 2 over expression (31), could be administered to treat steatosis or insulin resistance. In fact, PGE1 and PGE2 and their analogues are used in clinic to improve moderate hypercholesterolaemia among other liver pathologies (32). Furthermore, these results support caution with use of COX inhibitors (COXIBs) in patients with obesity, type 2 diabetes or NAFLD.
ACKNOWLEDGMENTS
The authors have not competing interests
Author contributions
D.E.F., O.M., N.A., A.G R., A. F A., C.C., E.G C., and A.M.V. researched the data.
L.C S. edited the manuscript.
L.B., C.E.C., R.M. and M.C. reviewed the manuscript and contributed to the discussion
A.M.V. and P.M S. wrote and reviewed the manuscript.
The authors thank to Gerardo Pisani (IFISE CONICET) Rosario, Argentina, for his technical assistance with the immunohistochemical analysis.
Dr. P. Martín Sanz and Dr. A.M. Valverde are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Page 19 of 47 Diabetes
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This work was supported by Financing Program for short stays abroad (CONICET
Argentina) to D.F.; L. C S is supported by a Post Doctoral fellowship from
CONACYT, México; SAF2012 39732 (MINECO, Spain) and CIBERehd (ISCIII,
Spain) to M.C.; BFU2011 24760 (MINECO, Spain) to L.B.; S2010/BMD 2378
(Comunidad de Madrid, CAM) to L.B. and P.M.S.; RD12/0042/0019 (ISCIII, Spain)
and CIBERehd (ISCIII, Spain) to L.B. and P.M.S; SAF2010 16037, SAF2013 43713 R
(MINECO, Spain) to P.M.S.; SAF2012 33283 (MINECO, Spain), S2010/BMD 2423
(Comunidad de Madrid, CAM), EFSD and Amylin Paul Langerhans Grant and
CIBERdem (ISCIII, Spain) to A.M.V.
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Table ΙΙΙ RCD HFD Wt hCOX 2 Tg Wt hCOX 2 Tg hCOX 2 Tg+DFU TG (mg/dl) 23.80 ± 2.70 23.66 ± 4.51 35.78 ± 3.08* 23.28 ± 1.89# 32.59 ± 1.63*† Cholesterol (mg/dl) 101.78 ± 12.98 72.71 ± 10.72 232.17 ± 25.68* 147.78 ± 9.13*# 207.90 ± 15.33*† NEFAs (mEq/l) 18.69 ± 2.21 16.79 ± 2.77 23.44 ± 2.96* 14.17 ± 2.43# 21.69 ± 4.42*† Insulin (ng/ml) 0.94 ± 0.30 0.72 ± 0.17 1.73 ± 0.36* 1.46 ± 0.13* 2.23 ± 0.66* Adiponectin (pg/ml) 7.21 ± 0.72 6.99 ± 1.64 6.08 ± 1.11 8.05 ± 0.35# 4.94 ± 0.61*† Leptin (pg/ml) 7.61 ± 2.07 3.82 ± 0.64* 24.93 ± 1.81* 21.29 ± 1.49*# 24.95 ± 0.89*† Adiponectin/Leptin Ratio 0.95 1.83* 0.24 0.38# 0.20 HOMA IR (A.U.) 19.01 ± 2.89 14.46 ± 0.92# 23.43 ± 0.63 # † PGE2 (pg/ml) 9.43 ± 1.16 14.18 ± 1.10* 8.98 ± 0.35 12.04 ± 0.48* 8.03 ± 0.34
Table I. Biochemical and metabolic characteristics of hCOX 2 Tg mice after HFD.
Plasma levels of triglycerides, cholesterol, non esterified free fatty acids (NEFAs) and
insulin, leptin and adiponectin from Wt, hCOX 2 Tg and hCOX 2 Tg+DFU mice fed
RCD and after 12 weeks fed HFD. Adiponectin/leptin ratio and HOMA IR. Plasma
PGE2 levels from Wt, hCOX 2 Tg and hCOX 2 Tg+DFU mice fed with RCD or HFD
diets. Data are expressed as means ± S.E. for 4 6 mice of each experimental group.
*p<0.05 vs. Wt RCD; #p<0.05 vs. Wt HFD, p<0.05 vs. hCOX 2 Tg HFD.
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Table ΙΙΙΙΙΙ
Gene RCD HFD Wt hCOX 2 Tg Wt hCOX 2 Tg hCOX 2 Tg+DFU Tnfa 1 0.65 ± 0.32 2.36 ± 0.79* 1.13 ± 0.38# 1.55 ± 0.66 Il1b 1 1.47 ± 0.30 3.16 ± 1.01* 1.52 ± 0.26# 1.47 ± 0.88 Il6 1 0.44 ± 0.10* 2.16 ± 0.68* 0.63 ± 0.20# 1.67 ± 0.81
Table II. Hepatic mRNA expression of pro inflammatory cytokines in hCOX 2 Tg
mice. Hepatic mRNA levels of pro inflammatory cytokines IL 6, IL 1β and TNF α
measured by real time q PCR normalized to the expression of 36b4 mRNA. Values
represent fold relative to Wt under RCD. Data are expressed as means ± S.E. for 4 6
mice of each experimental group. *p<0.05 vs. Wt RCD; #p<0.05 vs. Wt HFD.
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Table ΙΙΙΙΙΙΙΙ
Gene RCD HFD Wt hCOX 2 Tg Wt hCOX 2 Tg Cpt1a 1 1.15 ± 0.04 1.70 ± 0.20* 2.87 ± 0.27*# Pgc1a 1 0.68 ± 0.22 1.00 ± 0.13 0.99 ± 0.14 Acox1 1 0.73 ± 0.20 1.92 ± 0.24* 1.18 ± 0.39 Ppara 1 1.61 ± 0.18* 2.54 ± 0.10* 4.41 ± 0.14*# Pparg 1 0.16 ± 0.04* 5.95 ± 0.09* 2.94 ± 0.07*# Acaca 1 0.94 ± 0.18 1.21 ± 0.36 0.89 ± 0.16 Fasn 1 1.59 ± 0.43 1.32 ± 0.72 1.09 ± 0.35 Scd1 1 0.64 ± 0.24 2.25 ± 0.23* 1.46 ± 0.09*# Lipc 1 0.87 ± 0.17 1.05 ± 0.18 1.18 ± 0.16 Lipe 1 0.82 ± 0.08 1.70 ± 0.06* 1.80 ± 0.56 Pnpla2 1 0.97 ± 0.17 1.68 ± 0.56 2.55 ± 0.53 Cd36 1 0.67 ± 0.18 6.30 ± 0.38* 3.65 ± 0.22*# hcox2 1 7365.5 ± 453.8* 1.04 ± 0.43 6749.9 ± 266.2*# mpges1 1 1.58 ± 0.39 0.84 ± 0.35 1.68 ± 0.32*# CyclinD1 1 1.34 ± 0.08* 1.10 ± 0.21 1.51 ± 0.25*# Bcl2 1 1.13 ± 0.11 0.96 ± 0.20 1.57 ± 0.35*#
Table III. Hepatic mRNA expression of genes related to lipogenesis, lipolysis and β
oxidation in hCOX 2 Tg mice. Hepatic mRNA levels measured by real time q PCR
normalized to the expression of 36b4 mRNA, values represent fold relative to Wt under
RCD. Data are expressed as means ± S.E. for at 4 6 mice of each experimental group.
*p<0.05 vs. Wt RCD; #p<0.05 vs. Wt HFD.
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FIGURE LEGENDS
Fig. 1. hCOX 2 Tg mice are protected from HFD induced obesity, insulin and
glucose intolerances. Wt and hCOX 2 Tg mice were fed with HFD ad libitum for 12
weeks. An additional group of hCOX 2 Tg mice were treated with the COX 2 inhibitor
DFU five times in a week during all HFD treatment. (A) Representative photography of
Wt and hCOX 2 Tg mice. (B) Representative photography of eWAT and liver from Wt
and hCOX 2 Tg mice (C) Body weight curve of Wt, hCOX 2 Tg and hCOX 2
Tg+DFU mice fed a HFD expressed as % to basal body weight. Data are expressed as
means ± S.E. (n=7 per group). *p<0.05 vs. Wt; #p<0.05 vs. hCOX 2 Tg. (D) Initial (i)
and final (f) body weight and mean daily food intake. (E) Insulin Tolerance Test (ITT)
after 6 h fasting and (F) Glucose Tolerance Test (GTT) after 16 h fasting of animals
(n=6 per group) after HFD. Graph represents Area Under the Curve (AUC) during GTT.
Data are expressed as means ± S.E. *p<0.05 vs. Wt; #p<0.05 vs. hCOX 2 Tg.
Fig. 2. COX 2 transgenic mice are protected from HFD induced hepatic steatosis.
(A) Representative images of Hematoxylin & Eosin (H&E) stained liver paraffin
embedded sections from Wt and hCOX 2 Tg mice under RCD and from Wt, hCOX 2
Tg and hCOX 2 Tg+DFU mice under HFD. (B) Hepatic triglycerides levels in all
experimental groups, measured by enzymatic analysis. Data are expressed as means ±
S.E. (n=7 per group). *p<0.05 vs. Wt RCD; #p<0.05 vs. Wt HFD. (C) Representative
images of H&E